Process for preparing the releasing hormone of luteinizing hormone (LH) and of follicle stimulating hormone (FSH), salts and compositions thereof, and intermediates therefor

ABSTRACT

A process for preparing the LH- and FSH-releasing hormone of the formula I  H-Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2  which is isolated as the hydrochloride salt and optionally converted to other pharmaceutically acceptable salts or to pharmaceutically acceptable metal complexes.

This application is a continuation-in-part of each of application Ser.No. 243,465, filed April 12, 1972 (now abandoned) and of Ser. No.226,508, filed February 15, 1972, now U.S. Pat. No. 3,835,108, issuedSeptember 10, 1974.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention relates to a process for preparing the releasing hormoneof luteinizing hormone (LH) and of follicle stimulating hormone (FSH) inthe form of an acid addition salt, to salts thereof withpharmaceutically acceptable salts, to pharmaceutical compositionscontaining said LH and FSH-releasing hormone, and to intermediatesobtained in said process.

LH and FSH are both gonadotrophic hormones elaborated by the pituitarygland of humans and of animals. LH together with FSH stimulates therelease of estrogens from the maturing follicles in the ovary andinduces the process of ovulation in the female. In the male, LHstimulates the interstitial cells and is for that reason also calledinterstitial cell stimulating hormone (ICSH). The follicle-stimulatinghormone (FSH) induces maturation of the follicles in the ovary andtogether with LH, plays an important role in the cyclic phenomena in thefemale. FSH promotes the development of germinal cells in the testes ofthe male. Both LH and FSH are released from the pituitary gland by theaction of LH- and FSH-releasing hormone, and there is good evidence thatsaid releasing hormone is elaborated in the hypothalamus and reaches thepituitary gland by a neurohumoral pathway, see e.g. Schally et al.,Recent Progress in Hormone Research 24, 497 (1968).

The LH- and FSH-releasing hormone has been isolated from pig hypothalamiand its constitution elucidated by Schally et al., Biochem. Biophys.Res. Commun. 43, 393 and 1334 (1971), who proposed the decapeptidestructure

    (pyro) Glu -- His -- Trp -- Ser -- Tyr -- Gly -- Leu -- Arg -- Pro -- Gly -- NH.sub.2.

Thus, the hydroxyl group in serine does not have to be protected; theNH-groups in tryptophan and in histidine do not require protection; andno protection for the guanidino function in arginine and for thehydroxyl group of tyrosine is necessary in the later stages of theprocess. The process of this invention is thus also more convenient andless cumbersome than the processes of Prior Art. An added advantage ofthe process of this invention is the fact that the final step thereofconsists in the condensation of two unprotected fragments, each of whichis well defined, easy to purify, and each obtainable in a high state ofpurity. The final product thus obtained is the free, unprotecteddecapeptide which does not require any deprotective steps and isobtained in a high degree of purity and in good yields.

In the following text the term "lower alkyl" designates a straight orbranched chain alkyl group containing from 1-6 carbon atoms and includesmethyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, t-butyl and thelike. The term "lower" indicates 1-6 carbon atoms. The term "strongorganic base" denotes aliphatic and heterocyclic tertiary nitrogen basesand includes triethylamine, dibutylmethylamine, N-methylpyrrolidine,N-methylpiperidine, N-methylpiperazine, N-methylmorpholine and the like;triethylamine is preferred. The term "halogenated hydrocarbon" denotesthose having from 1-2 carbon atoms and includes methylene dichloride,ethylene dichloride, chloroform and the like; chloroform is preferred.The term "strong mineral acid" when used in conjunction with ananhydrous system, denotes hydrogen chloride, hydrogen bromide, andsulfuric acid; hydrogen chloride is preferred; when used in conjunctionwith an aqueous system the term includes any common mineral acid.

L-Pyroglutamic acid is the lactam of L-glutamic acid and has thestructure of 5-oxo-L-proline.

Many of the methods used in the syntheses of peptide linkages arecommonly designated by trivial names. Thus, the "azide method" comprisesthe reaction of an amino acid hydrazide having a suitably protectedamino group with a nitrite, usually t-butyl or isoamyl nitrite, toobtain the corresponding azide which is then reacted with an amino acidhaving a free amino and a suitably protected carboxylic acid group, toobtain the desired peptide.

The condensation with dicyclohexylcarbodiimide comprises the reaction ofan amino acid having a suitably protected amino and a free carboxylicacid group with another amino acid having a free amino and a suitablyprotected carboxylic acid group; the peptide linkage is formed withelimination of the elements of water and formation of dicyclohexylureawhich is easily removed from the reaction mixture. In the case where thefree amino group of the second amino acid reacts only with difficulty,for example if the free amino group is secondary such as in proline, itis advantageous to add hydroxysuccinimide to the reaction to form theintermediate hydroxysuccinimide ester of the first amino acid whichreacts readily with a secondary amino group to form the desired peptidelinkage. In principle, this modification of the dicyclohexylcarbodiimidemethod involves an activation of the carboxylic acid group, and suchactivation is also obtained when the 4-nitrophenyl or 2,4-dinitrophenylor 2,4,5-trichlorophenyl esters of the carboxylic acid are used insteadof the free acid. Such esters are generally known as activated esters.

The protective groups used in the process of this invention, and theconventional abbreviations by which they and the common amino acids aredesignated, are described in Schroder and Lubke, The Peptides, AcademicPress, New York and London 1965.

SUMMARY OF THE INVENTION

The process of this invention is summarily described in the followingsteps:

N-(5-Oxo-L-prolyl)-L-histidine hydrazide, obtained as described byGillessen et al., Helv. Chim. Acta 53, 63 (1970), is condensed by meansof the azide method with a lower alkyl or aralkyl ester of L-tryptophan,preferably the benzyl ester obtained as described by Wilchek et al., J.Org. Chem. 28, 1874 (1963), to yieldN-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophan benzyl ester. The latteris treated with hydrazine hydrate to yieldN-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophan hydrazide (II).

A lower alkyl ester of N-[O-benzyl-N-carboxy-L-tyrosyl]glycine N-benzylester, preferably the methyl ester prepared as described by Morley, J.Chem Soc. (C), 2410,(1967)is hydrolyzed to the corresponding free acidwhich is converted to the corresponding mixed anhydride with ethylchloroformate and treated with t-butyl carbazate to yieldN-[O-benzyl-N-carboxy-L-tyrosyl]glycine N-benzyl ester2-carboxyhydrazide t-butyl ester, and said last-named compound ishydrogenolyzed by means of hydrogen and a noble metal catalyst to yieldN-L-tyrosylglycine 2-carboxyhydrazide t-butyl ester. Condensation ofsaid last-named compound with an activated ester of N-carboxy-L-serylN-benzyl ester, preferably the 2,4-dinitrophenyl ester prepared asdescribed by Marchiori et al., Gazz. Chim. Ital., 93, 834 (1963) yieldsN-[N-(N-carboxy-L-seryl)-L-tyrosyl]glycine N-benzyl ester2-carboxyhydrazine t-butyl ester which is hydrogenolyzed as above toyield N-(N-L-seryl-L-tyrosyl)glycine 2-carboxyhydrazine t-butyl ester(III).

N-carboxy-L-proline N-benzyl ester, prepared as described by Berger etal., J. Am. Chem. Soc., 76, 5552 (1954) is condensed with a glycinelower alkyl ester, preferably the ethyl ester, usingdicyclohexylcarbodiimide as the condensing agent and the resultingproduct is treated with ammonia to obtain2-[(N-carboxy-L-prolyl)amino]acetamide N-benzyl ester. Said last-namedcompound is hydrogenolyzed and condensed with N-carboxy-N^(G)-nitro-L-arginine N-t-butyl ester, obtained as described by Hofmann etal., J. Am. Chem. Soc., 87, 620 (1965), using N-hydroxysuccinimide anddicyclohexylcarbodiimide as the condensing agents, to obtainN-[N-(N-carboxy-N^(G) -nitro-L-arginyl)-L-prolyl]glycinamide N-t-butylester (IV).

The same compound IV is also prepared by the following alternativeroute.

A lower alkyl ester of L-proline, preferably the methyl ester, andN-carboxy-N^(G) -nitro-arginine N-t-butyl ester, prepared respectivelyas described by Boissonas et al., Helv. Chim. Acta 44, 123 (1961) and byHofmann et al. cited above, are condensed by means ofdicyclohexylcarbodiimide to yield N-(N-carboxy-N^(G)-nitro-L-arginyl)-L-proline N-t-butyl ester. Said last-named compound iscondensed with a glycine lower alkyl ester, preferably the ethyl ester,using dicyclohexylcarbodiimide as the condensing agent and the resultingproduct is treated with ammonia to obtain N-[N-(N-carboxy-N^(G)-nitro-L-arginyl)-L-prolyl]glycinamide N-t-butyl ester (IV), identicalwith the product obtained as described above. The protectivet-butoxycarbonyl group of said last-named compound is removed bytreatment with acid and the resulting product is condensed with anactivated ester of N-carboxy-L-leucine N-benzyl ester, preferably the2,4,5-trichlorophenyl ester prepared as described by Kenner et al., J.Chem. Soc., 761 (1968), to yield N-[N-[N-(N-carboxy-L-leucyl)-N^(G)-nitro-L-arginyl]L-prolyl]glycinamide N-benzyl ester which ishydrogenolyzed in glacial acetic acid by means of hydrogen and a noblemetal catalyst to remove the protective nitro and carbobenzoxy groups toyield the corresponding diacetate salt,N-[N-[N-(N-L-leucyl)-L-arginyl]-L-prolyl]glycinamide diacetate (V).

N-[N-(5-Oxo-L-prolyl)-L-histidyl]-L-tryptophan hydrazide (II) andN-(N-L-seryl-L-tyrosyl)glycine 2-carboxyhydrazide t-butyl ester (III),both obtained as described above, are condensed by means of the azidemethod to yield the hexapeptideN-[N-[N-[N-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophyl]-L-seryl]-L-tyrosyl]glycine2-carboxyhydrazide t-butyl ester. The same compound is also obtained bycondensation of N-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophan, obtainedfrom the corresponding benzyl ester described above by hydrogenolysis,with N-(N-L-seryl-L-tyrosyl)glycine 2-carboxyhydrazide t-butyl ester(III), obtained as described above, using dicyclohexylcarbodiimide asthe condensing agent.N-[N-[N-[N-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophyl]-L-seryl]-L-tyrosyl]glycine2-carboxyhydrazide t-butyl ester obtained by either of the above routesis treated with trifluoroacetic acid to yield the trifluoroacetic acidsalt of the hexapeptide hydrazide, viz.N-[N-[N-[N-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophyl]-L-seryl]-L-tyrosyl]glycinehydrazide trifluoroacetate (VI).

Said last-named compound (VI) is condensed by means of the azide methodwith N-[N-[N-(N-leucyl)-L-arginyl]-L-prolyl]glycinamide diacetate (V) toyield the desired decapeptide5-oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosylglycyl-L-leucyl-L-arginyl-L-prolylglycinamide(I) which is isolated in the form of its hydrochloride salt. The lattersalt may be converted, if desired, into a different acid addition salt,e.g. a salt with a pharmaceutically acceptable acid, by treatment withthe appropriate ion exchange resin in the manner described by Boissonaset al., Helv. Chim. Acta 43, 1349 (1960). Suitable ion exchange resinsare strongly basic anion exchange resins, for example those listed inGreenstein and Winitz "Chemistry of the Amino Acids", John Wiley andSons, Inc., New York and London 1961, Vol. 2, p.1456. Basicallysubstituted cross-linked polystyrene resins such as Amberlite IRA-400 orIRA-410 are preferred. The above hydrochloride may also be converted toa salt of low solubility in body fluids by treatment with a slighlywater-soluble pharmaceutically acceptable acid.

Furthermore, the decapeptide of formula I may also be converted to itscomplexes with pharmaceutically acceptable metals. The acid additionsalts and the metal complexes of the LH- and FSH-releasing hormoneproduced by the process of this invention with pharmacologicallyacceptable acids or metals are biologically fully equivalent to thenatural hormone.

The above sequence of reactions, omitting the successive introductionand removal of protective groups as well as the methods of condensationand the condensing agents used, is shown below in FIG. 1.

                  FIG. 1                                                          ______________________________________                                        L-Pyroglutamic acid                                                           L-Histidine                                                                                             II.                                                 L-Tryptophan                                                                                                     VI.                                        L-Serine                                                                                                III.                                                L-Tyrosine                                                                     Glycine                                 I.                                   L-Leucine                                                                                                        V.                                         L-Arginine                                                                                      IV.                                                         L-Proline                                                                      Glycine                                                                        or                                                                          L-Arginine                                                                    L-Proline                                                                                       IV.                                                          Glycine                                                                      ______________________________________                                    

DETAILED DESCRIPTION OF THE INVENTION 1. LH- and FSH-Releasing Activity

The synthetic product of formula I obtained by the process of thisinvention in the form of an acid addition salt possesses LH- andFSH-releasing properties and is as active as the natural hormone whentested in the radioimmunoassay described by Niswender et al. Proc. Soc.Exp. Biol. Med., 128, 807 (1968). It is equally active in the assaydetermining induction of ovulation in the hamster described by Arimuraet al., Science 174, 511 (1971), and in a modification of the similarassay in the rat described by Arimura et al. in Endocrinology 80, 515(1967).

The LH- and FSH-releasing properties of the hormone obtained by theprocess of this invention, which in turn induce ovulation in animalsmake the hormone useful in veterinary practice and in animal husbandry.It is often desirable to synchronize estrus in livestock, for example,cattle, sheep, or swine either in order to be able to mate all thefemales in a given group with a male of the desired genetic quality, orso as to be able to perform artificial insemination on a maximum numberof females, both within a comparatively short period of time. In thepast, this has been done by administering to the animals anovulation-inhibiting agent, withdrawing administration of said agentshortly before the date chosen for mating or artificial insemination,and relying either upon the natural production of LH and FSH to induceovulation and to produce estrus or by administering gonadotrophins.However, this procedure was not entirely satisfactory because ovulationat a predetermined time occured never in all the animals together butonly in a certain proportion thereof when gonadotrophins were not used.On the other hand, the high cost of gonadotrophins and side effectsencountered in their administration made this method impractical. It isnow possible to obtain substantially complete synchronization ofovulation and of estrus, by treating the animals in a given group firstwith an ovulation inhibitor which is subsequently withdrawn, and thenadministering the LH- and FSH-releasing hormone produced by the processof this invention shortly before the predetermined period of time formating or artificial insemination, so as to obtain ovulation and estruswithin that time interval. The delay in the onset of ovulation andestrus following administration of the hormone produced by the processof this invention varies with the species of animal, and the optimaltime interval has to be chosen for each species. For example, in rodentssuch as rats or hamsters ovulation takes place within 18 hours followingadministration of the LH- and FSH-releasing hormone produced by theprocess of this invention.

The method described above for obtaining ovulation and estrus within aprecisely predetermined time interval, so as to be certain of asuccessful mating is particularly important for breeders of race horsesand of show animals, where the fees paid for the services of anexceptional male animal often amount to very considerable sums of money.

The LH- and FSH-releasing hormone produced by the process of thisinvention is also useful to increase the number of live births perpregnancy in livestock, for example, cattle, sheep or swine. For thispurpose the LH- and FSH-releasing hormone is given in a series ofparenteral doses, preferably by intravenous of subcutaneous injections,in the range of 1.0 mcg to 100 mcg per kilogram of body weight per day,96 to 12 hours prior to expected estrus and subsequent mating. A priminginjection of 1000 to 5000 IU of pregnant mares serum gonadotrophin mayalso be given 1 to 4 days prior to the above injection of LH- andFSH-releasing hormone. A similar treatment, with or without priorpriming, is also useful for inducing puberty in farm animals.

When the hormone produced by the process of this invention is employedfor the purpose of inducing ovulation and estrus or for inducing pubertyin warm-blooded animals, especially in rodents such as rats or hamstersor in livestock, it is administered systemically, preferablyparenterally, in combination with a pharmaceutically acceptable liquidor solid carrier. The proportion of the hormone is determined by itssolubility in the given carrier, by the chosen route of administration,and by standard biological practice. For parenteral administration toanimals the hormone may be used in a sterile aqueous solution which mayalso contain other solutes such as buffers or preservatives, as well assufficient pharmaceutically acceptable salts or glucose to make thesolution isotonic. The dosage will vary with the form of administrationand with the particular species of animal to be treated and ispreferably kept at a level of from 5 mcg to 100 mcg per kilogram bodyweight. However, a dosage level in the range of from about 10 mcg toabout 50 mcg per kilogram body weight is most desirably employed inorder to achieve effective results.

The hormone may also be administered in one of the long-acting,slow-release or depot dosage forms described below, preferably byintramuscular injection or by implantation. Such dosage forms aredesigned to release from about 0.5 mcg to about 50 mcg per kilogram bodyweight per day.

The LH- and FSH-releasing hormone produced by the process of thisinvention is also useful in human medicine. For example, human chorionicgonadotrophin (HCG) which contains mainly LH and some FSH has been usedfor over 30 years to treat certain endocrinological disorders such asdisturbances of the cycle, amenorrhea, lack of development of secondarysex characteristics, and infertility in the female, or certain cases ofhypogonadism, delayed puberty, cryptorchidism, and non-psychogenicimpotence in the male. Lately, infertility in the human female has alsobeen treated with human menopausal gondotrophin (HMG) which containsmainly FSH, followed by treatment with HCG. One of the disadvantages ofthe treatment of infertility in the human female with HCG or with HMGfollowed by HCG has become apparent in that such treatment often resultsin superovulation and unwanted multiple births, probably because of theimpossibility of giving only the exact amounts of FSH and LH which arenecessary for ovulation. The administration of the hormone produced bythe process of this invention overcomes the above disadvantage, becausethe hormone causes release of LH and FSH by the pituitary only in theexact quantities which are required for normal ovulation. For thatreason the hormone produced by the process of this invention is not onlyuseful for the above purpose, but it is equally useful in the humanfemale in the treatment of disturbances of the cycle, of amenorrhea, ofhypogonadism, and of lack of development of secondary sexcharacteristics.

Furthermore, the LH- and FSH-releasing hormone produced by the processof this invention is also useful in contraception. For example, when thehormone is administered to a human female early in the menstrual cycleLH is released at that time and causes premature ovulation. The immatureovum is either not capable of being fertilized, or, if fertilizationshould nevertheless have taken place, it is highly unlikely that thefertilized ovum will become implanted because the estrogen-progestinbalance required to prepare the endometrium is not present and theendometrium is not in the condition necessary for implantation. On theother hand, when the hormone is administered towards the end of thecycle the endometrium is disrupted and menstruation takes place.

In addition, the LH- and FSH-releasing hormone produced by the processof this invention is also useful in contraception by the "rhythm"method, which has always been relatively unreliable because of theimpossibility of predetermining ovulation in the human female with therequired degree of accuracy. Administration of the hormone at mid-cycle,i.e. at about the normally expected time for ovulation, inducesovulation shortly thereafter and makes the "rhythm" method both safe andeffective.

The LH- and FSH-releasing hormone produced by the process of thisinvention is also useful as a diagnostic tool for distinguishing betweenhypothalamic and pituitary malfunctions or lesions in the human female.When administering the hormone to a patient suspected of suchmalfunctions or lesions and a rise in the level of LH is subsequentlyobserved there is good indication to conclude that the hypothalamus isthe cause of the malfunction and that the pituitary is intact. On theother hand, when no rise in circulating LH is seen following theadministration of the hormone a diagnosis of pituitary malfunction orlesion can be made with a high degree of confidence.

In the human male, administration of the LH- and FSH-releasing hormoneobtained by the process of this invention provides the amounts of LH (orICSH) and of FSH necessary for normal sexual development in cases ofhypogonadism or delayed puberty, and is also useful in the treatment ofcryptorchidism. Furthermore, the FSH released by the administration ofthe hormone stimulates the development of germinal cells in the testes,and the hormone is useful in the treatment of non-psychogenic impotence.

When the LH- and FSH-releasing hormone obtained by the process of thisinvention in the form of an acid addition salt is employed in humanmedicine, it is administered systemically, either by intravenous,subcutaneous, or intramuscular injection, or by sublingual, nasal, orvaginal administration, in compositions in conjunction with apharmaceutically acceptable vehicle or carrier.

For administration by injection or by the nasal route as drops or sprayit is preferred to use the hormone in solution in a sterile aqueousvehicle which may also contain other solutes such as buffers orpreservatives, as well as sufficient quantities of pharmaceuticallyacceptable salts or of glucose to make the solution isotonic.

The LH- and FSH-releasing hormone produced by the process of thisinvention may also be administered as nasal or vaginal powders orinsufflations. For such purposes the hormone is administered in finelydivided solid form together with a pharmaceutically acceptable solidcarrier, for example a finely divided polyethylene glycol ("Carbowax1540"), finely divided lactose, or, preferably only for vaginaladministration, very finely divided silica ("Cab-O-Sil"). Suchcompositions may also contain other excipients in finely divided solidform such as preservatives, buffers, or surface active agents.

For sublingual or vaginal administration it is preferred to formulatethe hormone in solid dosage forms such as sublingual tablets or vaginalinserts or suppositories with sufficient quantities of solid excipientssuch as starch, lactose, certain types of clay, buffers, andlubricating, disintegrating, or surface-active agents, or withsemi-solid excipients commonly used in the formulation of suppositories.Examples of such excipients are found in standard pharmaceutical texts,e.g. in Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pennsylvania, 1970.

The dosage of the LH- and FSH-releasing hormone obtained by the processof this invention will vary with the form of administration and with theparticular patient under treatment. Generally, treatment is initiatedwith small dosages substantially less than the optimum dose of thehormone. Thereafter, the dosage is increased by small increments untilthe optimum effect under the circumstances is reached. In general, thehormone obtained by the process of this invention is most desirablyadministered at a concentration level that will generally affordeffective release of LH and of FSH without causing any harmful ordeleterious side effects, and preferably at a level that is in a rangeof from about 1 mcg to about 100 mcg per kilogram body weight, althoughas aforementioned variations will occur. However, a dosage level that isin the range of from about 5 mcg to about 50 mcg per kilogram bodyweight is most desirably employed in order to achieve effective results.

It is often desirable to administer the hormone continuously overprolonged periods of time in long-acting, slow-release, or depot dosageforms. Such dosage forms may either contain a pharmaceuticallyacceptable salt of the hormone having a low degree of solubility in bodyfluids, for example one of those salts described below, or they maycontain the hormone in the form of a water-soluble salt together with aprotective carrier which prevents rapid release. In the latter case, forexample, the hormone may be formulated with a non-antigenic partiallyhydrolyzed gelatin in the form of a viscous liquid; or the hormone maybe adsorbed on a pharmaceutically acceptable solid carrier, for examplezinc hydroxide, and may be administered in suspension in apharmaceutically acceptable liquid vehicle; alternatively, the hormonemay be administered in the form of a complex with a pharmaceuticallyacceptable metal for example zinc, copper, cobalt, iron, or aluminum;the zinc complex is preferred; or the hormone may be formulated in gelsor suspension with a protective non-antigenic hydrocolloid, for examplesodium carboxymethylcellulose, polyvinylpyrrolidone, sodium alginate,gelatine, polygalacturonic acids, for example, pectin, or certainmucopolysaccharides, together with aqueous or non-aqueouspharmaceutically acceptable liquid vehicles, preservatives, orsurfactants. Examples of such formulations are found in standardpharmaceutical texts, e.g. in Remington's Pharmaceutical Sciences citedabove. Long-acting, slow-release preparations of the hormone producedaccording to the process of this invention may also be obtained bymicroencapsulation in a pharmaceutically acceptable coating material,for example gelatine, polyvinyl alcohol or ethyl cellulose. Furtherexamples of coating materials and of the processes used formicroencapsulation are described by J. A. Herbig in Encyclopedia ofChemical Technology, Vol. 13, 2nd Ed., Wiley, New York 1967, pp.436-456. Such formulations, as well as suspensions of salts of thehormone which are only sparingly soluble in body fluids, are designed torelease from about 0.1 mcg to about 50 mcg of the hormone per kilogrambody weight per day, and are preferably administered by intramuscularinjection. Alternatively, some of the solid dosage forms listed above,for example certain sparingly water-soluble salts or dispersions in oradsorbates on solid carriers of salts of the hormones, for exampledispersions in a neutral hydrogel of a polymer of ethylene glycolmethacrylate or similar monomers cross-linked as described in U.S. Pat.No. 3,551,556 may also be formulated in the form of pellets releasingabout the same amounts as shown above and may be implantedsubcutaneously or intramuscularly.

Alternatively, slow-release effects over prolonged periods of time mayalso be obtained by administering the hormone obtained by the process ofthis invention as an acid addition salt in an intra-vaginal device or ina temporary implant, for example a container made of a non-irritatingsilicone polymer such as a polysiloxane, e.g. "Silastic", or of aneutral hydrogel of a polymer as described above, possessing therequired degree of permeability to release from about 0.1 mcg to about50 mcg per kilogram body weight per day. Such intra-vaginal or implantdosage forms for prolonged administration have the advantage that theymay be removed when it is desired to interrupt or to terminatetreatment.

2. Preparation of Compounds

The process of this invention is carried out in the following manner.

A solution of N-(5-oxo-L-prolyl)-L-histidine hydrazide (see Gillessen etal. cited above) in an inert anhydrous solvent, preferably a mixture ofdimethylformamide and dimethylsulfoxide, is cooled to a temperature offrom about -10° to about 5°C, mixed with a solution of a strong mineralacid, preferably hydrogen chloride, in an anhydrous ether or cyclicether, preferably tetrahydrofuran, and the mixture is cooled to atemperature of from about -30° to about -20°C. An organic nitrite,preferably t-butyl nitrite is isoamyl nitrite, is added with stirringand the mixture is stirred for 20-60 minutes, preferably for about 30minutes at a temperature of from about -30° to about -20°C. Sufficientquantities of a strong organic base, preferably triethylamine, are addedwith stirring to make the mixture alkaline, preferably pH 8-9. Keepingthe temperature of the mixture within the range of about -30° to about-10°C, a solution in an inert solvent, preferably dimethylformamide, ofa lower alkyl or aralkyl ester of L-tryptophan, preferably the benzylester (see Wilchek et al. cited above), is added in an amount of from5-15 per cent in excess over the molar equivalent. A molar excess ofabout 10 per cent is preferred, and the mixture is stirred at about -30°to about -10°C for 30-60 minutes and then in an ice bath for 16-24hours, preferably for about 18 hours. Filtration of the precipitate,evaporation of the filtrate, and crystallization of the residue yieldsthe corresponding N-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophan loweralkyl or aralkyl ester, the benzyl ester being preferred for purposes ofisolation and purification. Said last-named compound, preferably in theform of the benzyl ester, is dissolved in an anhydrous lower alkanol,preferably methanol, cooled to a temperature of from about -20° to about0°C, preferably about -10°C, a molar excess of hydrazine hydrate isadded with stirring and the mixture is stirred first at ice bathtemperature, for 2-6 hours, preferably for about 3 hours, and then for30-60 hours, preferably for about 40 hours, at room temperature(20°-25°C). The precipitate is filtered and crystallized from a loweralkanol, preferably methanol, to yieldN-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophan hydrazide (II.).

A lower alkyl ester of N-[O-benzyl-N-carboxy-L-tyrosyl]glycine N-benzylester, preferably the methyl ester (see Morley cited above) is dissolvedin a lower alkanol or alkoxyalkanol, preferably methoxyethanol, a molarexcess (5-20 per cent excess, preferably about 15 per cent excess) of anaqueous alkali metal hydroxide, preferably sodium hydroxide, is addedand the mixture is stirred at room temperature for 0.5-2 hours,preferably for about 1 hour. The mixture is acidified with a strongmineral acid, preferably hydrochloric acid, the precipitate is filteredand crystallized from an aqueous lower alkanol, preferably methanol -water, to yield N-[O-benzyl-N-carboxy-L-tyrosyl]glycine N-benzyl ester.Said last-named compound is dissolved in an anhydrous ether or cyclicether, preferably tetrahydrofuran, the solution is cooled to atemperature in the range of about -20° to about 0°C, preferably about-10°C, and substantially one molar equivalent of ethyl chloroformate isadded. The mixture is stirred at about -10°C for 5-30 minutes,preferably for about 10 minutes, and substantially one molar equivalentof t-butyl carbazate is added. The mixture is stirred first for 20-60minutes, preferably for about 30 minutes, at about 0°C and then forabout 3-8 hours, preferably for about 5 hours, at room temperature(20°-25°C). The solvent is evaporated, the residue taken up in asubstantially water-immiscible solvent, preferably a lower alkyl esterof a lower alkanoic acid, for example ethyl acetate, the solutionwashed, dried and evaporated, and the residue crystallized to yieldN-[O-benzyl-N-carboxy-L-tyrosyl]glycine N-benzyl ester2-carboxyhydrazide t-butyl ester. Said last-named compound is dissolvedin an anhydrous lower alkanol, preferably methanol, a noble metalcatalyst, e.g. palladium on charcoal, is added and the mixture isagitated in an atmosphere of hydrogen at room temperature untilsubstantially 2 molar equivalents of hydrogen have been taken up.Filtration of the catalyst and evaporation of the filtrate yields aresidue of N-L-tyrosylglycine 2-carboxyhydrazide t-butyl ester, usedwithout purification in the subsequent step.

Said last-named compound is dissolved in an anhydrous halogenatedhydrocarbon, e.g. chloroform, or preferably in dimethylformamide and theresulting solution is added, with exclusion of moisture, to a solutionof an activated ester of N-carboxy-L-seryl N-benzyl ester, preferablythe 2,4-dinitrophenyl ester (see Marchiori et al. cited above),previously cooled to about 0°C. The mixture is kept at about 0°C forseveral days, preferably for about 3 days, the solvent is evaporated,the residue taken up in a mixture of a halogenated hydrocarbon, a loweralkanol, and a small amount of a weak organic base, preferablychloroform-methanol-pyridine, and the solution is subjected tochromatography, preferably on silica. Elution, evaporation of theeluates, and crystallization yieldsN-[N-(N-carboxy-L-seryl)-L-tyrosyl]glycine N-benzyl ester2-carboxyhydrazide t-butyl ester. Said last-named compound is dissolvedin a lower alkanol, preferably methanol, a noble metal catalyst,preferably palladium on charcoal, is added, and the mixture is agitatedin an atmosphere of hydrogen at room temperature for 5-10 hours,preferably for about 7 hours until substantially one molar equivalent ofhydrogen has been taken up. Filtration of the catalyst, evaporation ofthe filtrate, and crystallization of the residue yieldsN-(N-L-seryl-L-tyrosyl)glycine 2-carboxyhydrazide t-butyl ester (III).

A solution of N-carboxy-L-proline N-benzyl ester (see Berger et al.cited above) and of an approximately equimolar amount of a glycine loweralkyl ester acid addition salt, preferably glycine ethyl esterhydrochloride, in a halogenated hydrocarbon solvent, preferablychloroform, is cooled to about 0°C and a substantially equimolar amountof a strong organic base, preferably triethylamine, followed by asubstantially equimolar amount of dicyclohexylcarbodiimide is added. Themixture is stirred at about 0°C for 12-24 hours, preferably for about 16hours, filtered, and the filtrate is washed, dried and evaporated. Theresidue is taken up in a lower alkanol saturated with ammonia at about0°C, preferably methanol, and the solution is allowed to stand at about0°C for 48-72 hours. Evaporation of the solvent and crystallizationyields 2-[(N-carboxy-L-prolyl)amino]acetamide N-benzyl ester. Saidlast-named compound is dissolved in glacial acetic acid, a noble metalcatalyst, preferably palladium on charcoal, is added and the mixture isagitated in an atmosphere of hydrogen until substantially one molarequivalent of hydrogen has been taken up. Filtration and evaporation ofthe filtrate yields 2-[(L-prolyl)amino]acetamide which is dissolved indimethylformamide containing a substantially equimolar amount of astrong organic base, preferably triethylamine, cooled and added at atemperature of from about -20° to about 0°C, preferably at about -10°C,to a solution of a N-carboxy-N^(G) -nitro-L-arginine N-lower alkylester, preferably the N-t-butyl ester (see Hofmann et al. cited above)in dimethylformamide containing about 2 molar equivalents ofN-hydroxysuccinimide and about one molar equivalent ofdicyclohexylcarbodiimide. The mixture is stirred at about -10° to about5°C, preferably at about 0°C, first for 12-24 hours at about 0°C andthen for 24-48 hours at room temperature (20°-25°C), evaporated, theresidue is taken up in a mixture of a halogenated hydrocarbon and alower alkanol, preferably chloroform and methanol, and is purified bychromatography on silica, to yield the corresponding N-lower alkyl esterof N-[N-(N-carboxy-N^(G) -nitro-L-arginyl)-L-prolyl]glycinamide,preferably the N-t-butyl ester (IV).

Alternatively, the same compound IV is also prepared as follows.

A lower alkyl ester acid addition salt of L-proline, preferably themethyl ester hydrochloride (see Boissonas et al. cited above), and anapproximately equimolar amount of N-carboxy-N^(G) -nitro-L-arginineN-t-butyl ester (see Hofmann et al. cited above) are dissolved in ananhydrous strongly polar solvent, for example dimethylsulfoxide,dimethylformamide, or acetonitrile, or mixtures thereof, preferably amixture of acetonitrile and dimethylformamide, and a substantiallyequimolar amount of a strong organic base, preferably triethylamine, isadded with stirring. The mixture is cooled to a temperature of fromabout -20° to about 0°C, preferably to about 10°C, a substantiallyequimolar amount of dicyclohexylcarbodiimide is added, the mixture isstirred at about 0°C for 2-10 hours, preferably for 5 hours, and thenfor 8-24 hours, preferably for about 10 hours, at room temperature(20°-25°C), filtered, and the filtrate evaporated. The residue is takenup in a halogenated hydrocarbon or in a substantially water-immisciblelower alkyl ester of a lower alkanoic acid, preferably chloroform orethyl acetate, washed, evaporated, the residue taken up in a loweralkanol, preferably methanol, and stirred at room temperature for about1-3 hours with about 2 molar equivalents of an aqueous alkali metalhydroxide, preferably sodium hydroxide. The mixture is filtered,extracted with a halogenated hydrocarbon or a lower alkyl loweralkanoate, preferably chloroform or ethyl acetate, the aqueous phase isacidified, extracted as above, and the extracts are evaporated to yieldN-(N-carboxy-N^(G) -nitro-L-arginyl)-L-proline N-t-butyl ester. Saidlast-named compound is dissolved in dimethylformamide, an acid additionsalt of a lower alkyl ester of glycine, preferably glycine ethyl esterhydrochloride, is added, the mixture is cooled to about 0°C, asubstantially equimolar amount of a strong organic base, preferablytriethylamine, and substantially one molar equivalent ofdicyclohexylcarbodiimide, are added and the mixture is stirred, first atabout 0°C for 2-5 hours, preferably for about 3 ours, and then at roomtemperature for 12-24 hours, preferably for about 17 hours. Filtration,evaporation of the solvent, taking up the residue in a mixture of ahalogenated hydrocarbon and a lower alkanol, preferably chloroform andmethanol, followed by chromatography on silica, evaporation of theeluates, taking the residue into a lower alkanol saturated with ammoniaat 0°C, preferably methanol, allowing the solution to stand at about 0°Cfor several days, preferably for about 3 days, evaporating the solventand crystallization yields N-[N-(N-carboxy-N^(G)-nitro-L-arginyl)-L-prolyl]glycinamide N-t-butyl ester (IV), identicalwith the product obtained by the different route described above.

Said last-named compound of formula IV, obtained by either of the routesdescribed above, is dissolved in a strong acid commonly used fordeprotection, preferably hydrogen chloride in a lower alkanol or inglacial acetic acid, or trifluoroacetic acid, the solution is kept atabout 0°C for 20-60 minutes, preferably for about 30 minutes, and isthen added to about 5-15 parts by volume, preferably about 10 parts pervolume, of an anhydrous inert water-immiscible ether-type solvent, suchas a lower alkyl ether, preferably diethyl ether, to yield a precipitateof the corresponding salt of N-[N-(N^(G)-nitro-L-arginyl)-L-prolyl]glycinamide. The latter is filtered,dissolved in dimethylformamide containing a substantially equimolaramount of a strong organic base, preferably triethylamine, the solutionstirred at about 0°C for 15-30 minutes, and a substantially equimolaramount of an activated ester of N-carboxy-L-leucine N-benzyl esterpreferably the 2,4,5-trichlorophenyl ester (see Kenner et al. citedabove) is added. The mixture is allowed to stand first at about 0°C for24-48 hours and then at room temperature for 24-48 hours. Evaporation ofthe solvent, dissolving the residue in a mixture of a halogenatedhydrocarbon and a lower alkanol, preferably chloroform-methanol,followed by chromatography on silica and evaporation of the eluateyields N-[N-(N-carboxy-L-leucyl)-N^(G)-nitro-L-arginyl]-L-prolyl]glycinamide N-benzyl ester. Said last-namedcompound is dissolved in glacial acetic acid, a noble metal catalyst,preferably palladium on charcoal, is added and the mixture is agitatedat room temperature in an atmosphere of hydrogen until substantially 4molar equivalents of hydrogen have been taken up. Filtration andevaporation of the solvent yieldsN-[N-[N-(N-L-leucyl)-L-arginyl]-L-prolyl]glycinamide diacetate (V) as anamorphous solid.

A solution of N-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophan hydrazide(II), obtained as described above, in an inert anhydrous solvent,preferably a mixture of dimethylformamide and dimethylsulfoxide, iscooled to a temperature of from about -10°C to about 5°C, mixed with asolution of a strong mineral acid, preferably hydrogen chloride, in ananhydrous ether or cyclic ether, preferably tetrahydrofuran, and themixture is cooled to a temperature of from about -30° to about -20°C. Anorganic nitrite, preferably t-butyl nitrite or isoamyl nitrite, is addedwith stirring in a substantially equimolar amount and the mixture isstirred for 20-60 minutes, preferably for about 30 minutes at atemperature of from about -30°C to about 20°C. Sufficient quantities ofa strong organic base, preferably triethylamine, are added with stirringto make the mixture alkaline, preferably pH 8-9. Keeping the mixture ata temperature of from about -30°C to about -10°C, a solution of asubstantially equimolar amount of N-(N-L-seryl-L-tyrosyl)glycine2-carboxyhydrazide t-butyl ester (III), obtained as described above, inan inert anhydrous solvent, preferably dimethylformamide, is added withstirring, and agitation is continued for 30-60 minutes at about -30°C toabout -10°C, then at about 0°C for another 30-60 minutes, and finallywith cooling in an ice bath for 16-24 hours, preferably for about 18hours. Filtration of the precipitate, evaporation of the filtrate,taking up the residue in a lower alkanol, preferably methanol, andprecipitation by addition of an ether, preferably diethyl ether,followed by crystallization from a lower alkanol, preferably methanol,yieldsN-[N-[N-[N-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophyl]-L-seryl]-L-tyrosyl]glycine2-carboxyhydrazide t-butyl ester.

Alternatively, that same compound may also be prepared as follows: Asolution of N-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophan benzyl ester,obtained as described above is prepared in a lower alkanol, preferablymethanol, or in glacial acetic acid, a noble metal catalyst, preferablypalladium on charcoal, is added and the mixture is agitated in anatmosphere of hydrogen at room temperature for 10-30 hours, preferablyfor about 16 hours, until substantially one molar equivalent of hydrogenhas been taken up. Filtration of the catalyst, evaporation of thefiltrate, taking up the residue in a lower alkanol, preferably methanol,precipitation by addition of an ether, preferably diethyl ether, andfiltration of the precipitate yieldsN-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophan, the same tripeptide ascompound II described above except that the terminal carboxylic acid isunsubstituted. Said last-named compound is dissolved in an inertanhydrous solvent, preferably dimethylformamide, a substantiallyequimolar amount of N-(N-L-seryl-L-tyrosyl)glycine 2-carboxyhydrazidet-butyl ester (III, obtained as described above) is added, the mixtureis cooled to about 0°C, a substantially one molar equivalent ofdicyclohexylcarbodiimide is added and the mixture is stirred, first atabout 0° C for about 24 hours, and then at room temperature for 4-10days, preferably for about 5 days. Filtration, evaporation of thesolvent, taking up the residue in a mixture of a halogenated hydrocarbonand a lower alkanol, preferably chloroform and methanol, followed bychromatography on silica and evaporation of the eluate, taking up theresidue in a lower alkanol, preferably methanol, and precipitation byaddition of an ether, preferably diethyl ether, followed by filtration,yieldsN-[N-[N-[N-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophyl]-L-seryl]-L-tyrosyl]glycine2-carboxyhydrazide t-butyl ester, identical with the same compoundprepared as described above.

Said last-named compound, prepared by either of the routes describedabove, is dissolved in trifluoroacetic acid, preferably of about 90%strength, and the solution is stirred for 30-60 minutes first withcooling in an ice bath and then for another 30-60 minutes at roomtemperature. Precipitation with an ether, preferably diethyl ether,followed by crystallization from a lower alkanol, preferably methanol,yields the hexapeptideN-[N-[N-[N-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophyl]-L-seryl]-L-tyrosyl]glycinehydrazide trifluoroacetate (VI).

Said last-named compound (VI) is dissolved in an inert anhydroussolvent, preferably a mixture of dimethylformamide anddimethylsulfoxide, and a solution of a strong mineral acid, preferablyhydrogen chloride, in an anhydrous ether or cyclic ether, preferablytetrahydrofuran, is added with stirring at a temperature of from -10°Cto 5°C, preferably at about 0°C. The mixture is cooled to a temperatureof from about -30°C to about -10°C, preferably to about -20°C, asolution of a substantially equimolar amount of an organic nitrite,preferably t-butyl nitrite or isoamyl nitrite, in dimethylformamide isadded and the mixture is stirred at about -30°C to about -10°C for 30-60minutes. A sufficient amount of a strong organic base, preferablytriethylamine, is added to make the mixture slightly alkaline,preferably pH 8-9, and a solution of a substantially equimolar amount ofN-[N-[N-(N-L-leucyl)-L-arginyl]-L-prolyl]glycinamide diacetate (V,obtained as described above) in an anhydrous inert solvent, preferablydimethylformamide, is added together with a quantity of a strong organicbase, preferably triethylamine, sufficient to neutralize the diacetatesalt. The mixture is stirred at a temperature of from about -30°C toabout -10°C for 30-60 minutes, then at about 0°C for another 30-60minutes, and finally with cooling in an ice bath for 16-24 hours.Filtration, evaporation of the filtrate, taking up the residue in alower alkanol, preferably methanol, and precipitation by addition on anether, preferably diethyl ether, yields the crude decapeptide which ispurified by partition chromatography on a chemically modifiedcross-linked dextran ("Sephadex LH-20") using the lower phase of an-butanolacetic acid-water mixture as solvent. Evaporation of theeluates, taking up the residue in a lower alkanol, preferably methanol,and precipitation by addition of an ether, preferably diethyl ether,yields the substantially pure decapeptide5-oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosylglycyl-L-leucyl-L-arginyl-L-prolyl-glycinamide(I), isolated as the hydrochloride salt which shows the same amino acidanalysis as the natural product and which is as potent as the latter inbioassays. If desired, the above hydrochloride salt may be convertedinto other acid addition salts, preferably those with pharmaceuticallyacceptable acids. For such purposes the above hydrochloride salt istreated with a strong basic anion exchange resin, for example one ofthose listed in Greenstein and Winitz "Chemistry of the Amino Acids",cited above, in the form of its salt with the acid of which it isdesired to form the salt with the hormone. Elution yields the LH- andFSH-releasing hormone in the form of its salt with the correspondingdesired acid. Preferred anion exchange resins are cross-linkedpolystyrene resins substituted with strongly basic groups such asAmberlite IRA-400 or IRA-410, and preferred acids are pharmaceuticallyacceptable acids, to obtain the corresponding pharmaceuticallyacceptable salts of the hormone.

If it is desired to obtain a salt of the decapeptide of formula I whichis sparingly soluble in water or in body fluids, an acid addition saltthereof as obtained by the process of this invention is treated inaqueous solution with a pharmaceutically acceptable sparinglywater-soluble acid, for example tannic, alginic, or pamoic acid,preferably in the form of one of their salts, for example the alkalimetal salts. The hormone precipitates as the salt with the respectivesparingly water-soluble acid and is isolated, for example by filtrationor centrifugation.

The above sequence of reactions constituting the process of thisinvention and incorporating only the first alternative syntheses of theintermediates IV and VI described above is shown in FIG. 2, using theconventional abbreviations for the various amino acids and protectivegroups.

                                      FIG. 2                                      __________________________________________________________________________    Pyr     His  Trp   Ser  Tyr Gly   Leu   Arg   Pro Gly                                                                              NH.sub.2                 __________________________________________________________________________    H       NHNH.sub.2                                                                         H OBz      Z   OMe               Z OH                                                                              H OEt                                                 Bz                                                  H              OBz      Z   OH                Z     OEt                                                 Bz                                                  H              NHNH.sub.2                                                                             Z   NHNHBoc           Z     NH.sub.2                                 (II.)      Bz                                                                     Z ODnp                                                                             H   NHNHBoc     Boc                                                                              OH H     NH.sub.2                                                             NO.sub.2                                              Z        NHNHBoc     Boc         NH.sub.2 (IV.)                                                       NO.sub.2                                              H        NHNHBoc                                                                              Z                                                                              OTcp                                                                               [H         NH.sub. 2 ].CF.sub.3                                                          COOH                                                  (III.)         NO.sub.2                           H                           NHNHBoc                                                                              Z                NH.sub.2                                                             NO.sub.2                           CF.sub.3 COOH[H             NHNH.sub.2 ]                                                                        [H                NH.sub.2 ].2CH.sub.3                                                          COOH (V.)                                             (VI.)                                             [H                                                  NH.sub.2 ].2H.sub.2                                                           O.HCl                     __________________________________________________________________________

The decapeptide prepared according to the process of this invention mayalso be converted to a sparingly soluble complex thereof with apharmaceutically acceptable metal. Such complexes are prepared bydissolving the decapeptide or a water-soluble salt thereof in an aqueoussolution of a salt of said metal and slowly raising the pH of themixture by addition of a water-soluble base, for example sodiumhydroxide, until substantially all of the decapeptide has precipitatedas the metal complex, and isolating said metal complex by filtration orcentrifugation. The zinc complex is preferred and is pharmacologicallyequivalent to the decapeptide.

The following Examples will illustrate the invention. All compounds areidentified by elementary analysis.

EXAMPLE 1 N-[N-(5-Oxo-L-Prolyl)-L-Histidyl]-L-Tryptophan Benzyl Ester

N-(5-Oxo-L-prolyl)-L-histidine hydrazide (0.840 g, 3 mmol) is dissolvedin a solution at 0°C of dry dimethylformamide (14 ml), dry dimethylsulfoxide (10.5 ml) and 2.4 N anhydrous gaseous hydrogen chloride in drytetrahydrofuran (7.5 ml). The solution is cooled to -20°C and isoamylnitrite (0.57 ml) is added with stirring. The solution is stirred for 30minutes at -20°C and is then cooled to -24°C. Triethylamine (3.2 ml) isslowly added until the solution is slightly alkaline pH 8-9. Whilestirring at -20°C, a solution of L-tryptophan benzyl ester (0.970 g, 3.3mmol) in dry dimethylformamide (3.0 ml) is added. The solution isstirred at -20°C for 30 minutes, at 0°C for 30 minutes, and then at icebath temperature for 18 hours. The solution is filtered and theprecipitate is washed with dry dimethylformamide (2 × 2 ml). Thecombined filtrates are concentrated under reduced pressure at 40°C. Theresidue is dissolved in methanol (10 ml), diethyl ether (300 ml) isslowly added, and the precipitate is collected by filtration. Theprecipitate is once more dissolved in methanol (10 ml) and isprecipitated by the addition of diethyl ether (300 ml). The precipitateis crystallized from methanol to give the title compound as a fine whitepowder, m.p. 250°-252°C, [α]_(D) ²⁵ +1.6° (c = 1.0, DMF).

In the same manner, when using t-butyl nitrite instead of isoamylnitrite, or when using the methyl, ethyl, isopropyl or n-butyl ester oftryptophan, the corresponding methyl, ethyl, isopropyl or n-butyl esterof the title compound is also obtained.

EXAMPLE 2 N-[N-(5-Oxo-L-Prolyl)-L-Histidyl]-L-Tryptophan Hydrazide (II)

Hydrazine hydrate (99%, 1.5 ml) is slowly added to a stirred solution ofN-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophan benzyl ester (Example 1,0.868 g) in anhydrous methanol (150 ml) at -10°C. After stirring for 3hours at ice bath temperature the solution is stirred for 40 hours atroom temperature. The mixture is filtered and the crystallineprecipitate is recrystallized from methanol to give the title compoundas fine needles, m.p. 165°-169°C, [α]_(D) ²⁵ -24.6° (c = 1.0, DMF).

In the same manner, when using the methyl, ethyl, isopropyl, or n-butylester of the starting material instead of the benzyl ester, the titlecompound is also obtained.

EXAMPLE 3 N-[N-(5-Oxo-L-Prolyl)-L-Histidyl]-L-Tryptophan

A mixture of N-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophan benzyl ester(Ex. 1, 0.300 g, 0.555 mmol) and 5 percent palladium on carbon (0.060 g)in glacial acetic acid (30 ml) is stirred rapidly under an atmosphere ofhydrogen for 16 hours at room temperature. The mixture is filteredthrough diatomaceous earth ("Celite") and the filtrate is concentratedunder reduced pressure. The residue is dissolved in methanol (10 ml) anddiethyl ether (100 ml) is slowly added with vigorous stirring. Theprecipitate is collected, dissolved in methanol (10 ml) and diethylether (100 ml) is slowly added. The precipitate is collected, dissolvedin methanol, treated with charcoal (0.20 g) and filtered throughdiatomaceous earth ("Celite"). The filtrate is evaporated to yield thetitle compound as a hard white glass which is used as such in asubsequent step.

EXAMPLE 4 N-[O-Benzyl-N-Carboxy-L-Tyrosyl]Glycine N-Benzyl Ester

To a solution of N-[0-benzyl-N-carboxy-L-tyrosyl]glycine N-benzyl estermethyl ester (2 g, 0.0042 moles) in methoxyethanol (25 ml) aqueoussodium hydroxide (1N, 5 ml) is added with vigorous stirring. Whencomplete solution of the precipitate is achieved (after about 1 hour),aqueous hydrochloric acid (1N, 5.25 ml) is added with cooling. Theprecipitate is washed with water and recrystallized from methanol-waterto give the title compound with m.p. 166°-167°C, [α]_(D) ²⁵ -22.6° (c =1.0, DMF).

In the same manner, when using the ethyl, propyl, isopropyl or n-butylester of the starting material instead of the methyl ester, or whenusing methanol, ethanol, propanol, butanol, or 2-ethoxyethanol("Cellosolve") as the solvent instead of methoxyethanol, the titlecompound is also obtained.

EXAMPLE 5 N-[O-Benzyl-N-Carboxy-L-Tyrosyl]Glycine N-Benzyl Ester2-Carboxyhydrazide t-Butyl Ester

A solution of N-[O-benzyl-N-carboxy-L-tyrosyl]glycine N-benzyl ester(Example 4, 2.312 g, 0.005 moles) in dry tetrahydrofuran (12.5 ml) andtriethylamine (0.7 ml, 0.005 moles) is cooled to -10°C. With stirring,ethyl chloroformate (0.48 ml, 0.005 moles) and after 10 minutes t-butylcarbazate (725 mg, 0.005 moles) are added and the mixture is kept for 30minutes at 0°C and for 5 hours at room temperature. After evaporation ofthe solvent the residue is taken up in ethyl acetate (100 ml), filteredand the filtrate washed successively with water (2 × 20 ml), aqueousammonia (3 × 20 ml) and saturated sodium chloride solution (2 × 25 ml).The ethyl acetate layer is dried, evaporated under reduced pressure andthe residue crystallized from benzene-hexane to give the title compoundwith m.p. 88°-90°C.

EXAMPLE 6 N-L-Tyrosylglycine 2-Carboxyhydrazide t-Butyl Ester

To a solution of N-[O-benzyl-N-carboxy-L-tyrosyl]-glycine N-benzyl ester2 -carboxyhydrazide t-butyl ester (Example 5, 2.88 g, 0.005 moles) indry methanol (40 ml) palladium on charcoal (5%, 200 mg) is added. Thehydrogenation is carried out with the hydrogenation vessel connected toa stirred solution of sodium hydroxide (4N, 40 ml) to absorb carbondioxide. After 4 hours the theoretical amount of hydrogen is consumed.The catalyst is filtered off through diatomaceous earth ("Celite"), thefiltrate evaporated under reduced pressure at 30°C and the titlecompound obtained as an oily residue which is used without purificationin the next step.

In the same manner, when using platinum instead of palladium as acatalyst, the title compound is also obtained.

EXAMPLE 7 N-[N-(N-Carboxy-L-Seryl)-L-Tyrosyl]Glycine N-Benzyl Ester2-Carboxyhydrazide t-Butyl Ester

To a solution of N-carboxy-L-seryl N-benzyl ester 2,4-dinitrophenylester (1.6 g, 0.004 moles) in dry dimethylformamide (10 ml) cooled to0°C and protected from moisture a solution of N-L-tyrosylglycine2-carboxyhydrazide t-butyl ester (Example 6, 1.689 g, 0.005 moles) indry dimethylformamide is added. The solution is kept at 0°C for 3 days.After evaporation of the solvent the residue is taken up inchloroform-methanol-pyridine (100:25:1) and chromatographed over a 100fold amount of silica. The pure material is taken into a small amount ofmethanol and precipitated by addition of ether to give the pure titlecompound with m.p. 65°-95°C, [α]_(D) ²⁵ -13.1° (c = 1.0, DMF).

EXAMPLE 8 N-(N-L-Seryl-L-Tyrosyl)glycine 2-Carboxyhydrazide t-ButylEster (III)

A mixture of N-carboxy-L-seryl-L-tyrosylglycine N-benzyl ester2-carboxyhydrazide t-butyl ester (Example 7, 0.96 g) and 5 % palladiumon carbon (0.090 g) in methanol (35 ml) is rapidly stirred under anatmosphere of hydrogen for 7 hours at room temperature in the samemanner as described in Example 6 (hydrogen absorbed 49 ml). The mixtureis filtered through diatomaceous earth ("Celite") and the filtrate isconcentrated under reduced pressure. The residue is crystallized frommethanol-diethyl ether to give the title compound as fine white prismswith m.p. 132°-135°C, [α]_(D) ²⁵ -5.4° (c = 1.0, DMF).

In the same manner, but using platinum instead of palladium as acatalyst, the title compound is also obtained.

EXAMPLE 9 2-[(N-Carboxy-L-Prolyl)Amino]Acetamide) N-Benzyl Ester

To a stirred solution of N-carboxy-L-proline N-benzyl ester (12.5 g,0.05 moles) and glycine ethyl ester hydrochloride (7.26 g, 0.052 moles)in chloroform (100 ml) at 0°C, triethylamine (7.28 ml, 0.052 moles)followed by dicyclohexylcarbodiimide (10.7 g, 0.052 moles) is added.After stirring for 16 hours at 0°C the precipitate is filtered off andthe filtrate washed successively with water (50 ml), hydrochloric acid(1N, 50 ml), water (50 ml), and saturated sodium chloride solution (50ml), dried with magnesium sulfate and evaporated. The residue is takenup in methanol saturated with ammonia at 0°C (150 ml) and left at thistemperature for 48 hours. After evaporation of the solvent under reducedpressure the residue is recrystallized from methanol-ether to yield thetitle compound with m.p. 112°-115°C, further recrystallized frommethanol-isopropyl ether to m.p. 116°-120°C, [α] _(D) ²⁵ -43.8° (c =1.0, MeOH)

In the same manner, but using the methyl, propyl, isopropyl, or n-butylester of glycine instead of the ethyl ester, the title compound is alsoobtained.

EXAMPLE 10 2-[(L-Prolyl)Amino]Acetamide Hydrochloride

A solution of 2-[(N-carboxy-L-prolyl)amino]acetamide N-benzyl ester (Ex.9, 610 mg, 2 mmoles) in glacial acetic acid (8 ml) containing hydrogenchloride (2 mmoles) is agitated with palladium on charcoal (5%, 50 mg)in an atmosphere of hydrogen in the same manner as described in Example6 until one molar equivalent of hydrogen has been consumed. Filtration,evaporation of the filtrate and drying of the residue under reducedpressure gives the title compound which is used as such in thesubsequent step.

In the same manner, but using platinum instead of palladium as acatalyst, the title compound is also obtained.

Also in the same manner, but using hydrogen bromide or sulfuric acidinstead of hydrogen chloride, the corresponding hydrobromide or sulfatesalts of the title compound are obtained.

EXAMPLE 11 N-[N-(N-Carboxy-N^(G) -Nitro-L-Arginyl)-L-Prolyl]GlycinamideN-t-Butyl Ester (IV)

To a solution of N-carboxy-N^(G) -nitro-arginine N-t-butyl ester (607mg, 1.9 mmole) and N-hydroxysuccinimide (437 mg, 3.8 mmole) indimethylformamide (5 ml) at -10°C dicyclohexylcarbodiimide (388 mg., 1.9mmoles) is added. The mixture is stirred 1 hour at -10°C, 2 hours at 0°Cand 2 hours at room temperature. 2-[(L-Prolyl)amino]acetamidehydrochloride obtained as the residue in Example 10 is dissolved indimethylformamide (4 ml) at 0°C, triethylamine (0.38 ml) is added andthis solution is added to the solution of the active ester describedabove at about 0°C. The mixture is stirred for 14 hours at 0°C and for24 hours at room temperature, then evaporated under reduced pressure todryness. The residue is taken up in chloroform-methanol (100:15) andchromatographed over 100 g of silica. The pure title compound separatesfrom absolute ethanol as a gelatinous precipitate, sintering at about165°C, [α]_(D) ²⁵ -25.80°(c = 1.0, DMF).

In the same manner, but using the hydrobromide or sulfate salts of2-[(L-prolyl)amino]acetamide instead of the hydrochloride salt, thetitle compound is obtained.

EXAMPLE 12 N-(N-Carboxy-N^(G) -Nitro-L-Arginyl)-L-Proline N-t-ButylEster

To a solution of L-proline methyl ester hydrochloride (5.85 g, 35.4mmoles) and N-carboxy-N^(G) -nitro-arginine N-t-butyl ester (9.4 g, 29.4mmoles), in acetonitrile (10 ml) and dimethylformamide (25 ml)triethylamine (4.95 ml) is added. The mixture is cooled to -10°C anddicyclohexylcarbodiimide (7.25 g, 35.2 mmoles) is added. After 5 hoursat 0°C and 10 hours at room temperature the mixture is filtered and thefiltrate evaporated to dryness under reduced pressure. The resultingresidue is taken in ethyl acetate (300 ml) and washed successively withcitric acid solution (10%, 2 × 90 ml) ammonia solution (1N, 3 × 90 ml),and saturated sodium chloride solution (90 ml). The oily residue leftafter drying and evaporation of the organic layer is taken in methanol(88 ml). Aqueous sodium hydroxide solution (1N, 88 ml) is added and themixture is stirred for one hour at room temperature. After filtrationthe filtrate is extracted with ethyl acetate (3 × 150 ml). The aqueousphase is acidified with hydrochloric acid (4N, 30 ml) and extracted withethyl acetate (3 × 150 ml). The combined ethyl acetate extracts arewashed with saturated sodium chloride solution, dried with magnesiumsulfate and evaporated under reduced pressure. The title compound isobtained as an amorphous residue which shows one main spot inelectrophoresis in an aqueous buffer at pH 5.3 containing pyridine (1%)and acetic acid (0.32%) at 4,000 volt. It is used without furtherpurification in the next step.

In the same manner, when using the ethyl, isopropyl, or n-butyl ester ofL-proline instead of the metyl ester, the title compound is alsoobtained.

EXAMPLE 13 N-[N-(N-Carboxy-N^(G) -Nitro-L-Arginyl)-L-Prolyl]GlycinamideN-t-Butyl Ester (IV)

N-(N-Carboxy-N^(G) -nitro-L-arginyl)-L-proline N-t-butyl ester (Ex. 12,7.11 g, 17.1 mmoles) and glycine ethyl ester hydrochloride (2.5 g, 17.1mmoles) are dissolved in dimethylformamide (45 ml). With stirring at 0°Ctriethylamine (2.4 ml) and after 10 minutes dicyclohexylcarbodiimide(3.52 g, 17.1 mmoles) are added. The mixture is stirred for 3 hours at0°C and for 17 hours at room temperature, then filtered and the filtrateevaporated to dryness under reduced pressure. After chromatography onsilica (500 g) with chloroform-methanol (100:8) as solvent andevaporation an oily product is obtained. It is dissolved in methanolsaturated with ammonia at 0°C and left at this temperature for 3 days.The solvents are evaporated and the residue triturated with ethylacetate. The resulting title compound precipitates from absolute ethanolas a gelatinous product with m.p. 153°-163°C, [α]_(D) ²⁵ -26° (c = 1.0,DMF), identical with the same compound obtained as described in Example11.

In the same manner, when using the methyl, isopropyl, or n-butyl esterof glycine instead of the ethyl ester, the title compound is alsoobtained.

EXAMPLE 14 N-[N-[N-(N-Carboxy-L-Leucyl)-N^(G)-Nitro-L-Arginyl]-L-Prolyl-]Glycinamide N-Benzyl Ester

A solution of N-[N-[(N-carboxy-N^(G)-nitro-L-arginyl)-L-prolyl]glycinamide t-butyl ester (Exs. 11 or 13, 817mg, 1.76 mmoles) in trifluoroacetic acid (5 ml) is kept for 30 minutesat 0°C. The solution is added dropwise to cold absolute ether (50 ml),the precipitate of N-[N-(N^(G) -nitro-L-arginyl)-L-prolyl]glycinamidetrifluoroacetate is filtered off and washed well with dry ether. Afterdrying under reduced pressure the solid is taken in dimethylformamide (6ml) containing triethylamine (0.245 ml) and stirred for 15 minutes at0°C. To this solution a solution of N-carboxy-L-leucine N-benzyl ester2,4,5-trichlorophenyl ester (860 mg, 1.96 mmoles) and a drop of aceticacid are added and the mixture is left for 24 hours at 0°C and for 24hours at room temperature. After evaporation to dryness under reducedpressure the residue is taken up in chloroform-methanol (100:15) andchromatographed over 150 g of silica. The chromatographically pure titlecompound is obtained as an amorphous solid, dissolved in methanol (3 ml)and precipitated by adding this solution to cold isopropyl ether (50ml), to give the title compound with [α]_(D) ²⁵ -38.4° (c = 1.0, DMF).

In the same manner as described above, but using instead oftrifluoroacetic acid hydrogen chloride in a lower alkanol or in glacialacetic acid, the hydrochloride of N-[N-(N^(G)-nitro-L-Arginyl)-L-prolyl]glycinamide is obtained and is converted tothe title compound in the same manner as described above.

In the same manner the title compound is also obtained when the4-nitrophenyl or 2,4-dinitrophenyl esters of N-carboxy-L-leucine N-butylester are used instead of the 2,4,5-trichlorophenyl ester.

EXAMPLE 15 N-[N-[N-(N-L-Leucyl)-L-Arginyl]-L-Prolyl]GlycinamideDiacetate (V)

A solution of N-[N-[N-(N-carboxy-L-leucyl)-N^(G)-nitro-L-arginyl]-L-prolyl]glycinamide N-benzyl ester (Ex. 14, 805 mg,1.3 mmoles) in glacial acetic acid (5 ml) containing palladium oncharcoal catalyst (5%, 300 mg) is agitated under hydrogen in the samemanner as described in Example 6. The reaction mixture is filteredthrough diatomaceous earth ("Celite"), the filtrate concentrated underreduced pressure and added dropwise with stirring to dry ether at 0°C.The amorphous precipitate is filtered, washed well with ether and driedunder reduced pressure, to give the title compound which is homogeneousin thin layer chromatography on silica using ethyl acetate-aceticacid-n-butanol-water (1:1:1:1) as the solvent, and by electrophoresis atpH 1.6 in 8% aqueous formic acid at 4,000 volt or at pH 5.3 in anaqueous buffer containing 1% pyridine and 0.32% acetic acid at 4,000volt.

In the same manner, when using platinum instead of palladium as acatalyst, the title compound is also obtained.

EXAMPLE 16N-[N-[N-[N-[N-(5-Oxo-L-Prolyl)-L-Histidyl]-L-Tryptophyl]-L-Seryl]-L-Tyrosyl]glycine2-Carboxyhydrazide t-Butyl Ester

Method A

N-[N-(5-Oxo-L-prolyl)-L-histidyl]-L-tryptophan hydrazide (II., Ex. 2,0.655 g, 1.40 mmole) is dissolved at 0°C in a solution of drydimethylformamide (7 ml), dry dimethyl sulfoxide (5.9 ml) and 2.4 Nanhydrous gaseous hydrogen chloride in dry tetrahydrofuran (3.5 ml). Thesolution is cooled to -20°C and isoamyl nitrate (0.206 ml, 1.53 mmol) isadded with stirring. The solution is stirred for 30 minutes at -20°C andis cooled to -25°C. Triethylamine (1.3 ml) is slowly added until thesolution is slightly alkaline, pH 8-9. While stirring at -20°C, asolution of N-(N-L-seryl-L-tyrosyl)glycine 2-carboxyhydrazide t-butylester (III., Ex. 8, 0.617 g, 1.40 mmol) in dry dimethylformamide (6ml)is added. The solution is stirred at -20°C for 30 minutes at 0°C for 30minutes, and at ice bath temperature for 18 hours. The solution isfiltered and the precipitate is washed with dry dimethylformamide (2 × 2ml). The combined filtrates are concentrated under reduced pressure at40°C. The residue is dissolved in methanol (5 ml), diethyl ether (500ml) is slowly added, and the precipitate is collected by filtration. Theprecipitate is dissolved in methanol (5 ml) and is precipitated byaddition of diethyl ether (500 ml). The collected precipitate iscrystallized from methanol to give the title compound as a fine whitepower with m.p. 182°C (dec).

In the same manner, when using t-butyl nitrite instead of isoamylnitrite, the title compound is also obtained.

Method B

N-[N-(5-oxo-L-prolyl)-L-histidyl]-L-trptophan (Ex. 3, 0.133 g, 0.294mmol) and N-(N-L-seryl-L-tyrosyl)glycine 2-carboxyhydrazide t-butylester (III, Ex. 8, 0.129 g, 0.294 mmol) are dissolved in anhydrousdimethylformamide (5 ml). With stirring at 0°C, dicyclohexylcarbodiimide(0.061 g, 0.294 mmol) is added. The solution is stirred for 24 hours at0°C and for 5 days at room temperature, then filtered and the filtrateevaporated to dryness under reduced pressure. After chromatography onsilica (20 g) with chloroform-methanol (2:1) as solvent the combinedfractions are concentrated under reduced pressure. The residue isdissolved in methanol (2 ml), diethyl ether (100 ml) is slowly added,and the precipitate is collected by filtration. The resulting product isidentical with the title compound obtained as described above.

EXAMPLE 17N-[N-[N-[N-[N-(5-Oxo-L-Prolyl)-L-Histidyl]-L-Tryptophyl]-L-Seryl]-L-Tyrosyl]glycineHydrazide Trifluoroacetate (VI)

A solution ofN-[N-[N-[N-[N-(5-oxo-L-prolyl)-L-histidyl]-L-tryptophyl]-L-seryl]-L-tyrosyl]glycine2-carboxyhydrazide t-butyl ester (Example 16, 0.859 g, 0.982 mmol) in 90percent trifluoroacetic acid (27 ml) is stirred at ice bath temperaturefor 30 minutes and then at room temperature for 30 minutes. The solutionis slowly added to a stirred solution of diethyl ether (700 ml). Theprecipitate is collected and crystallized from methanol to yield thetitle compound as a fine white powder with mp 200°C (dec.), [α]_(D) ²⁵-21.2° (c = 1.0, water).

EXAMPLE 185-Oxo-L-Prolyl-L-Histidyl-L-Tryptophyl-L-Seryl-L-Tyrosylglycyl-L-Leucyl-L-Arginyl-L-Prolyglycinamide(I) Dihydrate Hydrochloride

N-[N-[N-[N-[N-(5-Oxo-L-prolyl)-L-histidyl]-L-tryptophyl]-L-seryl]-L-tyrosyl]glycinehydrazide trifluoroacetate (VI, Ex. 17, 0.840 g, 0.762 mmol) isdissolved at 0°C in a mixture of dry dimethylformamide (3.7 ml), drydimethylsulfoxide (3.3 ml) and 1.65 N anhydrous gaseous hydrogenchloride in dry tetrahydrofuran (2.77 ml). The solution is cooled to-20°C and a 10% solution of isoamyl nitrite in dry dimethylformamide(1.12 ml, 0.83 mmol) is added with stirring. The solution is stirred for30 minutes at -20°C and then cooled to -25°C. Triethylamine (0.7 ml) isslowly added until the solution is slightly alkaline, pH 8 - 9. Whilestirring at -20°C, a solution ofN-[N-[N-(N-L-leucyl)-L-arginyl]-L-prolyl]glycinamide diacetate (V,Example 15, 0.427 g, 0.762 mmol) in dry dimethylformamide (4.5 ml) andtriethylamine (0.28 ml) is added. The resulting solution is stirred at-20°C for 30 minutes, at 0°C for 30 minutes and at ice bath temperaturefor 18 hours. The solution is filtered and the precipitate is washedwith dry dimethylformamide (2 × 2 ml). The combined filtrates areconcentrated under reduced pressure at 40°C. The residue is dissolved inmethanol (5 ml), and diethyl ether (500 ml) is slowly added. Theprecipitate is collected, dried and purified by partition chromatographyon a chemically modified cross-linked dextran ("Sephadex LH-20") usingthe lower phase of n-butanol-acetic acid-water (8:4:40). The combinedfractions are concentrated to dryness under reduced pressure at 45°C,dissolved in methanol (5 ml), and added to diethyl ether (250 ml). Theprecipitate is collected by filtration and dried to yield the titlecompound with [α]_(D) ²⁵ -53.50° (c = 1.0, 1% aqueous acetic acid),isolated as the hydrochloride salt.

Calc. for C₅₅ H₇₅ N₁₇ O.sub. 13 . 2 H₂ O . HCL:

C: 52.67; H: 6.43; N: 18.99; O: 19.14; Cl: 2.83 %; found C: 52.25; H:6.23; N: 19.32; O: 19.10; Cl: 2.95%.

When carrying out the precipitation described above under strictlyanhydrous conditions and in the presence of hydrogen chloride, the titlecompound is obtained as the dihydrochloride salt.

Calc. for C₅₅ H₇₅ N₁₇ O₁₃ . 2 HCl:

C: 52.62; H: 6.18; N: 18.96; O: 16.57; Cl: 5.64 %; found C: 52.10; H:5.92; N: 18.85; O: 17.20; Cl: 5.75%.

Amino acid analysis gives the following composition:

    Histidine                                                                             1.0      1.04     Proline                                                                              0.96   1.03                                  Arginine                                                                              0.95     0.93     Glycine                                                                              2.01   1.98                                  Serine  0.90     0.86     Leucine                                                                              0.86   1.02                                  Glutamic                                                                              1.07     1.04     Tyrosine                                                                             1.12   1.04                                  Acid                                                                      

Electrophoresis at pH 1.6 in 8% aqueous formic acid and at 3500 voltgives a single spot and shows the uniformity of the compound.

In the same manner, when using t-butyl nitrite instead of isoamylnitrite, the title compound is also obtained.

In the same manner, but using other inorganic acids such as hydrogenbromide or sulfuric acid in tetrahydrofuran instead of hydrogenchloride, the hydrobromide or sulfate salts of the title compound arerespectively obtained.

If desired, the above hydrochloride salt is treated with AmberliteIRA-400 or IRA-410, previously converted to a salt thereof with apharmaceutically acceptable acid. Elution yields the corresponding saltof the title compound. Alternatively, the above hydrochloride salt istreated in aqueous solution with an alkali metal salt of tannic,alginic, or pamoic acid and the corresponding tannate, alginate orpamoate salt of the hormone is isolated by filtration or centrifugation.

EXAMPLE 195-Oxo-L-Prolyl-L-Histidyl-L-Tryptophyl-L-Seryl-L-Tyrosylglycyl-L-Leucyl-L-Arginyl-L-Prolylglycinamide(I) Zinc Complex

The dihydrochloride salt of the decapeptide of formula 1 obtained asdescribed in Example 18 (100 mg) is dissolved in water (0.8 ml) at roomtemperature and a zinc acetate solution [0.19 ml, Zn(OCOCH₃)₂ .2H₂ O,0.1 g/ml] is added. A 2N NaOH solution (0.1 ml) is added to the mixturedropwise under stirring. Every drop of sodium hydroxide causes formationof a precipitate (in lumps) which is stirred until it disappears and aclear solution is again obtained. The process is completed by additionof 0.1 N NaOH solution until the pH rises to 8.0-8.2 when theprecipitate can not be dissolved anymore. The end of precipitation ischecked by addition of one drop of NaOH solution to the clearsupernatant. After standing in the refrigerator overnight the titlecompound is separated by filtration, washed with water (0.1 ml) anddried in a high vacuum, uv(E+OH) = 289 nm (E_(1cm).^(1%) 42), 279 nm(E_(1cm).^(1%) 56) and 222 nm (E_(1cm).^(1%) 374).

We claim:
 1. A diagnostic method for distinguishing between hypothalmicand pituitary malfunctions or lesions in humans which comprisesadministering systemically to a human suspected of such malfunction fromabout 1 to 100 micrograms per kilogram of body weight of5-Oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosylglycyl-L-leucyl-L-arginyl-L-prolylglycinamideor a pharmaceutically acceptable salt thereof, and detecting the rise inthe level of serum leutenizing hormone.
 2. The diagnostic method ofclaim 1 wherein5-Oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosylglycyl-L-leucyl-L-arginyl-L-prolylglycinamidedihydrochloride is employed and the human is female.